Nucleic acid purification method

Abstract

The present invention relates to a method for purifying a defined amount of nucleic acids from a nucleic acid-containing sample, which has at least the following steps: (a.) contacting the nucleic acid-containing sample with a defined amount of a nucleic acid binding phase with the following features: (i) the nucleic acid binding phase has nucleic acid binding ligands that have at least one protonatable group; (ii) the nucleic acid binding ligands are bound to a carrier; (iii) the nucleic acid binding phase has a surface with a low charge density, wherein the amount of nucleic acids in the sample exceeds the binding capacity of the amount of nucleic acid binding phase used; (b.) binding of the nucleic acids to the nucleic acid binding phase at a pH (binding pH) that is below the pKs value of at least one of the protonatable groups; (c.) elution of the nucleic acids at a pH that is above the binding pH, wherein a defined amount of nucleic acids is obtained. Furthermore, corresponding kits and nucleic acid binding phases, which can be used for the purification of nucleic acids, are disclosed.

The invention claimed is: 
     
       1. A method for isolating and/or purifying a defined amount of nucleic acids from a nucleic acid-containing sample, the method having at least the following steps:
 a. contacting the nucleic acid-containing sample with a defined amount of a nucleic acid binding phase with the following features:
 (i) the nucleic acid binding phase has nucleic acid binding ligands, which have at least one protonatable group; 
 (ii) the nucleic acid binding ligands are bound to a carrier, wherein the low charge density is achieved by at least one of the following features:
 (aa) the nucleic acid binding ligands have, bound to the carrier, in each case not more than one or two protonatable groups for binding of the nucleic acids, and/or 
 (bb) the nucleic acid binding ligands are selected from the group of mono- and diamines, and/or 
 (cc) the carrier is coated with a mixture of nucleic acid binding ligands and diluting groups, wherein the proportion of nucleic acid binding ligands relative to the diluting groups is ≦50%; and/or 
 (dd) the nucleic acid binding ligands bound to the carrier are in deficit, such that ≦50% of functional groups or groups functionalizable with nucleic acid binding ligands on the carrier are functionalized with nucleic acid binding ligands; 
 
 (iii) the nucleic acid binding phase has a surface with a low charge density, wherein the amount of nucleic acids in the sample exceeds the binding capacity of the amount of nucleic acid binding phase used; 
 
 b. binding of the nucleic acids to the nucleic acid binding phase at a pH which is a binding pH that is below the pKs value of at least one of the protonatable groups; 
 c. elution of the nucleic acids at a pH that is above the binding pH, wherein a defined amount of nucleic acids is obtained. 
 
     
     
       2. The method according to  claim 1 , wherein at least one of said protonatable group(s) have at least one of the following features:
 a. a pKs value from 9 to 12; 
 b. a pKs value from 10 to 12; 
 c. the protonatable groups are amino groups and are not conjugated with the electron density-lowering groups. 
 
     
     
       3. The method according to  claim 1 , wherein the nucleic acid binding phase has at least one of the following features:
 a. the carrier and/or the nucleic acid binding ligands have functional groups that promote the release of the nucleic acids at the elution pH, optionally cation exchangers as functional groups, and optionally acid groups which are optionally carboxyl groups; 
 b. the nucleic acid binding ligands are selected from the group consisting of primary, secondary and tertiary amines of the formulas
 R 3 N, R 2 NH, RNH 2  and/or X—(CH 2 ) n —Y 
 with 
 X=R 2 N, RNH or NH 2    
 Y=R 2 N or RNH or NH 2    
 R=independently of one another, a linear, branched or cyclic alkyl, alkenyl, alkynyl or aromatic substituent, wherein the latter can also contain one or more heteroatoms; 
 n=0 to 20; 
 
 c. the carrier has no nucleic acid binding properties of its own apart from the nucleic acid binding ligands; 
 d. the carrier is selected from the group consisting of polystyrene and derivatives thereof, polyacrylates and polymethacrylates and derivatives thereof, polyurethanes, nylon, polyethylene, polypropylene, polybutylene and copolymers of these materials, agarose, cellulose, dextran, Sephadex, Sephacryl, chitosan, inorganic carriers, glass, metal oxides and metalloid oxides, Al 2 O 3 , TiO 2 , silica, boron oxide, carriers with metal surfaces, gold and magnetic particles and mixtures thereof, tubes, membranes, fleece, paper, reaction vessels, multiplates, chips and microarrays; and/or 
 e. the binding capacity is in the range from 0.1 to 500 μg nucleic acid per mg nucleic acid binding phase. 
 
     
     
       4. The method according to  claim 1 , wherein binding takes place under conditions that have at least one of the following features:
 a. binding takes place at a pH from 3 to 8; and/or 
 b. binding takes place at a pH from 4 to 7.5; and/or 
 c. binding takes place at a pH from 4.5 to 7; and/or 
 d. binding takes place at a pH from 5.5 to 7; and/or 
 e. binding takes place at a pH from 6.5 to 7; and/or 
 f. binding takes place at a salt concentration of ≦1M, ≦0.5M, ≦0.25M or ≦0.1M. 
 
     
     
       5. The method according to  claim 1 , wherein elution takes place under conditions that have at least one of the following features:
 a. elution takes place at a pH that is above the binding pH, but at least one pH unit below the pKs value of at least one of the protonatable groups; and/or 
 b. elution takes place at a pH from 7.5 to 10; and/or 
 c. elution takes place at a pH from 8 to 9; and/or 
 d. elution takes place at a pH from 8.2 to 8.8; and/or 
 e. elution takes place at a salt concentration of ≦1M, ≦0.5M, ≦0.25M, ≦0.1M, ≦25 mM, ≦15 mM or ≦10 mM; and/or 
 f. for elution, a solution is used that is selected from the group of water, biological and organic buffer. 
 
     
     
       6. The method according to  claim 1 , wherein a washing step is carried out after binding and prior to elution. 
     
     
       7. The method according to  claim 6 , wherein a washing solution is used that has at least one of the following features:
 a. water or an aqueous solution with low salt concentration is used for the washing step; and/or 
 b. an aqueous solution with low salt concentration is used, wherein the salt concentration is ≦400 mM, ≦200 mM, ≦100 mM, ≦50 mM and/or ≦25 mM. 
 
     
     
       8. A nucleic acid binding phase recitable for isolating and/or purifying a defined amount of nucleic acids from a nucleic acid-containing sample, wherein the nucleic acid binding phase has nucleic acid binding ligands with at least one protonatable group, wherein the nucleic acid binding ligands are bound to a carrier and the nucleic acid binding phase has a surface with a low charge density, wherein the low charge density is achieved by at least one of the following features:
 a. the nucleic acid binding ligands have, bound to the carrier, in each case not more than one or two protonatable groups for binding the nucleic acids; and/or 
 b. the nucleic acid binding ligands are selected from the group of mono- and diamines; and/or 
 c. the carrier is coated with a mixture of nucleic acid binding ligands and diluting groups, wherein the proportion of nucleic acid binding ligands relative to the diluting groups is ≦50%; and/or 
 d. the nucleic acid binding ligands bound to the carrier are in deficit, such that ≦50% of functional groups or groups functionalizable with nucleic acid binding ligands on the carrier are functionalized with nucleic acid binding ligands; 
 
       wherein an elution pH is established that is above the binding pH and wherein the amount of nucleic acid binding phase is selected so that the nucleic acids in the sample are in excess to the binding capacity of the nucleic acid binding phase used. 
     
     
       9. The nucleic acid binding phase according to  claim 8 , wherein the nucleic acid binding phase has at least one of the following features:
 a. the protonatable group(s) have a pKs value from 9 to 12; and/or 
 b. the protonatable groups are amino groups and are not conjugated with the electron density-lowering groups; and/or 
 c. the nucleic acid binding phase has functional groups that promote the release/elution of the nucleic acids at the elution pH, the functional groups optionally being cation exchangers, optionally being carboxyl groups; and/or 
 d. the binding capacity of the nucleic acid binding phase is in the range from 0.1 to 500 μg nucleic acid per mg nucleic acid binding phase; 
 e. the carrier has no nucleic acid binding properties of its own apart from the nucleic acid binding ligands; and/or 
 f. the carrier is selected from the group consisting of polystyrene and derivatives thereof, polyacrylates and polymethacrylates and derivatives thereof, polyurethanes, nylon, polyethylene, polypropylene, polybutylene and copolymers of these materials, agarose, cellulose, dextran, Sephadex, Sephacryl, chitosan, inorganic carriers, glass or further metal oxides and metalloid oxides, Al 2 O 3 , TiO 2 , silica, boron oxide, carriers with metal surfaces, gold, magnetic particles or mixtures thereof, tubes, membranes, fleece, paper, reaction vessels, multiplates, chips and microarrays; and/or 
 g. the nucleic acid binding ligands are selected from the group consisting of primary, secondary and tertiary amines of the formulas
 R 3 N, R 2 NH, RNH 2  and/or X—(CH 2 ) n —Y 
 with 
 X=R 2 N, RNH or NH 2    
 Y=R 2 N or RNH or NH 2    
 R=independently of one another, a linear, branched or cyclic alkyl, alkenyl, alkynyl or aromatic substituent, wherein the latter can also contain one or more heteroatoms; 
 n=0 to 20. 
 
 
     
     
       10. A kit for isolating and/or purifying a defined amount of nucleic acids from a nucleic acid-containing sample, wherein said kit comprises a nucleic acid binding phase as defined in  claim 8 . 
     
     
       11. The kit according to  claim 10 , comprising at least one of the following features:
 a. a binding buffer or a binding solution with a pH that is at least one pH unit below the pKs value of at least one of the protonatable groups of the nucleic acid binding phase, and/or a binding buffer or a binding solution that enables the adjustment of said pH in the sample; and/or 
 b. an elution buffer or an eluting solution with a pH that is one pH unit below the pKs value of at least one of the protonatable groups of the nucleic acid binding phase, and/or an elution buffer or an eluting solution that enables the adjustment of said pH in the sample. 
 
     
     
       12. The kit according to  claim 10 , wherein
 a. the binding buffer or the binding solution has at least one of the following features:
 i. a pH from 3 to 8; and/or 
 ii. a pH from 4 to 7.5; and/or 
 iii. a pH from 4.5 to 7; and/or 
 iv. a pH from 5.5 to 7; and/or 
 v. a pH from 6.5 to 7; and/or 
 vi. a salt concentration of ≦1M, ≦0.5M, ≦0.25M or ≦0.1M; 
 
 
       and/or
 b. the elution buffer or the eluting solution has at least one of the following features:
 i. a pH from 7.5 to 10; and/or 
 ii. a pH from 8 to 9; and/or 
 iii. a pH from 8.2 to 8.8; and/or 
 iv. a salt concentration of ≦1M, ≦0.5M, ≦0.25M, ≦0.1M, ≦25 mM, ≦15 mM or ≦10 mM; and/or 
 v. it is selected from the group consisting of water, biological and organic buffer. 
 
 
     
     
       13. The method of  claim 9 , wherein the nucleic acid binding phase is a polymer particle modified with N-propyl-1,3-propanediamine. 
     
     
       14. The method according to  claim 1 , wherein the method is carried out following a classical nucleic acid purification technique, in order to normalize already purified nucleic acids and obtain a defined amount of nucleic acids. 
     
     
       15. The method according to  claim 1 , wherein the normalized nucleic acid is used for an enzymatic reaction, a modification reaction, or a sequencing reaction. 
     
     
       16. The method of  claim 15 , wherein said enzymatic reaction is a nucleic acid amplification reaction. 
     
     
       17. The method according to  claim 1 , wherein the nucleic acid is a PCR fragment.